Fig. 5
RING-induction by the toxin drives senescence and infection. a Representative image of toxin-induced cell growth arrest and distension. HT1080s either untreated or treated with toxinWT or toxinHQ for 2 h before imaging at 7-days. Cell colonies (top panel) and high-magnification images (bottom panel). Arrows mark individual cells. Scale bars 50 μm. b Representative image of SA-β-Gal activity in intoxicated cells. Scale bars 50 μm. c Proportion of intoxicated cells with SA-β-Gal activity relative to untreated. Analysed 1700 cells/variable, error bars SD. d Representative image of toxin-induced SA-β-Gal in γH2AX RING cells at 48 h. Blue arrows indicate senescent RING cells (γH2AX in magenta/SPiDER β-Gal in grayscale) and distended nuclei (DAPI, greyscale). White arrows indicate non-senescent cells. Scale bars 50 μm. Proportion of cells with SA-β-Gal activity following (e) intoxication with toxinWT ± serum (350 cells/variable, error bars SD), f RPA knockdown (900 cells/variable, error bars SEM), g APH-treatment (900 cells/variable, error bars SD), and h infection with wild-type (SmWT) or toxin-deficient (SmΔcdtB) S.Javiana (3500 cells/variable, error bars SEM). i Experimental pipeline for j, k and l assaying toxin-induced transmissible senescence. Conditioned medium harvested from cells 24 h post-treatment, as indicated, was incubated with fresh cells for 6-days before assaying: j SA-β-Gal activity in HT1080s (1240 cells/variable, error bars SD). k γH2AX (green) positive HT1080 cell nuclei (DAPI, blue). One biological replicate, 300 cells/variable, error bars SD. Representative γH2AX images shown left. Scale bars 50 μm. l Salmonella infection of THP1s. 370 cells/variable, error bars SD. Representative images of THP1s (DAPI, blue) infected with SalmonellaΔcdtB (green), left. Scale bars 50 μm. In graphs, coloured circles indicate means from technical replicates (two biological replicates, unless indicated otherwise). Statistical significance (**** = P < 0.0001, *** = P < 0.0002, ** = P < 0.0021, * = P < 0.0332, ns = P > = 0.05) was calculated relative to control (indicated by lack of p-value) using a one-way ANOVA and a Tukey’s multiple comparison test (c, g, j, k, l), or an unpaired two-sided t test (e, f, h). Source data are provided as a Source Data file
